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Abstract:

The invention relates to genes coding for TCP family transcription
factors and having a biological role in the development of axillary buds
and branch growth. Furthermore, the invention relates to the promoters of
the transcription of said genes, to the genetic constructs containing
same and to the uses thereof, including the use of agents that modulate
the expression of these genes in order to modify plant architecture.

41. An expression modulating agent of the polynucleotide according to
claim 40, wherein said agent is: a) an antisense oligonucleotide, an
interfering RNA, or an antibody or a fragment thereof, or b) interfering
RNA having the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13.

42. An isolated regulatory expression sequence of a gene of interest in
the axillary meristem of a plant, wherein: a) the regulatory expression
sequence comprises at least 95% identity with the nucleotide sequence of
SEQ ID NO: 5 or SEQ ID NO: 6, or b) the regulatory expression sequence is
SEQ ID NO: 5 or SEQ ID NO: 6.

43. A genetic construction comprising: (a) a polynucleotide capable of
being translated into an amino acid sequence comprising: (i) a peptide
having at least 95% identity with the amino acid sequence SEQ ID NO: 2 or
SEQ ID NO: 3, or (ii) the peptide SEQ ID NO: 2 or SEQ ID NO: 3; (b) an
expression modulating agent of the polynucleotide according to (a),
wherein said agent is: (i) an antisense oligonucleotide, an interfering
RNA, or an antibody or a fragment thereof, or (ii) interfering RNA having
the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13; or (c) a
regulatory expression sequence operatively linked to the polynucleotide
according to (a) or the expression modulating agent according to (b),
wherein: (i) the regulatory expression sequence comprises at least 95%
identity with the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or
(ii) the regulatory expression sequence is SEQ ID NO: 5 or SEQ ID NO: 6.

44. A plant comprising the genetic construction according to claim 43 (a)
or (c), wherein the regulatory expression sequence in (c) is operatively
linked to the polynucleotide according to (a).

46. The plant according to claim 44, which can be taxonomically
classified as belonging to the species Solanum lycopersicum or Solanum
tuberosum.

47. A plant which can be taxonomically classified as belonging to the
species Solanum lycopersicum or Solanum tuberosum having a plant
architecture modified with respect to a control-type plant, wherein the
modification of the plant architecture is due to non-transgenic mutations
in any polynucleotide according to claim 40.

48. A seed, a plant cell, a part of the plant or a grain of pollen of the
plant according to claim 44.

49. A method to modify the architecture of a plant, reducing the number
of branches in respect to a control plant, using the polynucleotide
according to claim 40, a genetic construction comprising the
polynucleotide according to claim 40, or a genetic construction
comprising a regulatory expression sequence operatively linked to the
polynucleotide according to claim 40, wherein the regulatory expression
sequence comprises: (i) at least 95% identity with the nucleotide
sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or (ii) the regulatory
expression sequence is SEQ ID NO: 5 or SEQ ID NO: 6.

50. A method to modify the architecture of a plant, increasing the number
of branches in respect to a control plant, using the expression
modulating agent according to claim 41, a genetic construct comprising
the expression modulating agent according to claim 41, or a genetic
construct comprising a regulatory expression sequence operatively linked
to the expression modulating agent according to claim 41, wherein the
regulatory expression sequence comprises: (i) at least 95% identity with
the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or (ii) the
regulatory expression sequence is SEQ ID NO: 5 or SEQ ID NO: 6.

51. A method to modify the plant architecture of a plant, reducing the
number of branches in respect to a control plant, comprising: (a)
transfecting a polynucleotide according to claim 40, a genetic
construction comprising the polynucleotide according to claim 40, or a
genetic construction comprising a regulatory expression sequence
operatively linked to the polynucleotide according to claim 40 in a cell
or culture of host plant cells, wherein the regulatory expression
sequence comprises: (i) at least 95% identity with the nucleotide
sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or (ii) the regulatory
expression sequence is SEQ ID NO: 5 or SEQ ID NO: 6; and (b) growing the
transfected cell or culture of host plant cells in a suitable medium,
until regenerating a complete plant.

52. A method to modify the plant architecture of a plant, increasing the
number of branches in respect to a control plant, comprising: (a)
transfecting the expression modulating agent according to claim 41, a
genetic construct comprising the expression modulating agent according to
claim 41, or a genetic construct comprising a regulatory expression
sequence operatively linked to the expression modulating agent according
to claim 41 in a cell or host plant cell culture, wherein the regulatory
expression sequence comprises: (i) at least 95% identity with the
nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or (ii) the
regulatory expression sequence is SEQ ID NO: 5 or SEQ ID NO: 6; and (b)
growing the transfected cell or culture of host plant cells in a suitable
medium, until regenerating a complete plant.

53. The method according to claim 51, wherein the transfected cell can be
taxonomically classified as belonging to the species Solanum lycopersicum
or Solanum tuberosum.

54. A method for obtaining the plant according to claim 47, comprising:
(a) obtaining plant material from a plant (parent) which can be
taxonomically classified as belonging to the species Solanum lycopersicum
or Solanum tuberosum; (b) subjecting the plant material of step (a) to a
mutagenesis process; (c) culturing the mutated plant material until
regenerating a complete plant, and its descendants; (d) analysing the
descendants of the plants of step (c) to detect at least one mutation in
at least one copy of any polynucleotide according to claim 40, (e)
selecting the descendants with at least one mutation in at least one copy
of the polynucleotide according to claim 40, which have their plant
architecture modified in comparison with a control plant; and (f)
optionally, culturing the plant selected to obtain descendants which has
said modification of the plant architecture.

55. The plant according to claim 45, which can be taxonomically
classified as belonging to the species Solanum lycopersicum or Solanum
tuberosum.

56. A seed, a plant cell, a part of the plant or a grain of pollen of the
plant according to claim 45.

57. A seed, a plant cell, a part of the plant or a grain of pollen of the
plant according to claim 46.

58. A seed, a plant cell, a part of the plant or a grain of pollen of the
plant according to claim 47.

59. The method according to claim 52, wherein the transfected cell can be
taxonomically classified as belonging to the species Solanum lycopersicum
or Solanum tuberosum.

Description:

[0001] The present invention belongs to the field of molecular biology,
biotechnology and plant improvement, and specifically relates to genes
coding for transcription factors of the TCP family and having a
biological role in the development of the axillary buds and branch
growth. It also relates to the transcription promoters of said genes, to
the genetic constructs containing same and to the uses thereof, including
the use of agents that modulate the expression of these genes, to modify
the plant architecture.

PRIOR ART

[0002] One of the central questions in biology is the effect of the
evolution of genomes in morphological diversity. The body plans are
determined by the genetic routes of development widely conserved in large
taxonomic groups. Changes in the activity of the genes that control these
pathways give rise to alterations in the morphological templates. Most of
these changes are deleterious, but a few may give rise to evolution in
the form. In angiosperm plants the branching templates are determined by
the position in which the branches are formed. The branches are generated
from meristems formed in the axillary buds of the leaves after
germination of the seeds. The axillary meristems (AM) give rise to
axillary buds, structures containing preformed branches with short
internodes, leaf primordia, new AM and, often, floral meristems. The buds
can remain inactive during long periods of time or sprout giving rise to
branches due to elongation of the internodes, in response to
environmental or endogenous signals. This decision determines the plant
architecture and affects key aspects of plant life, such as the amount of
nutrients each growth axis will receive, plant height, the sun protection
of the fruit, efficiency in light absorption or their visibility for
pollenizers.

[0003] The genes that control the start of the AM, the development of the
buds and their sprouting have been characterized in different species of
angiosperms. These studies indicate that the development of the axillary
buds is controlled by conserved genetic routes which evolved before the
radiation of the plants with flowers. The start of AM is controlled by
the genes Ls/LAS/MONOCULM1 and the genes Blind/RAX1 in tomatoes (Solanum
lycopersicum), Arabidopsis, and rice. Auxin and strigolactone, hormones
synthesized in the apices of the shoots and in the root, respectively,
promote long-distance signalling to suppress branching in various
species. The synthesis and the response to the strigolactones through the
conserved pathway MAX/RMS, described in Arabidopsis and pea, have also
been found in petunia monocotyledon (Petunia hybrida), and rice. The
genes which act within the buds delaying their development and growth are
also conserved. The gene Teosinte branched1 (Tb1) isolated in corn and in
other monocotyledon codes for a transcription factor of the TCP family.
The TCP genes, exclusive of plants, code for transcription factors
containing the so-called TCP domain, a sequence of 59 amino acids with a
basic region and a helix-loop-helix domain, which gives DNA linking
capacity and to other proteins (Cubas et al., 1999. Plant Journal.
18:215-222), which is expressed in the AM and in the axillary buds, where
its growth is suppressed. Tb1 also controls the flowering and the
development of inflorescence. In dicotyledon, the duplication of Tb1 has
given rise to three types of genes (CYC1, CYC2 and CYC3) one of which,
CYC1-type, seems to have retains the majority of the branching
suppression activity, at least in Arabidopsis, where this gene receives
the name of BRANCHED1 (BRC1). BRC1 acts within the buds preventing their
development. BRC1 is controlled transcriptionally by the MAX route and
responds to environmental and developmental stimuli suppressing the
branching.

[0004] Despite the fact that the genes having a key role in the control of
axillary development are very conserved, the diversity of the branching
models found in angiosperms suggest that the modulation of this process
has diverged into different phylogenetic groups (clades), which is
supported by the different regulation of the genes of MAX type in pea,
Arabidopsis and rice. It is very possible that the function and
regulation evolution of the BRC1 type genes have also played an important
role in this evolution. Unlike the alterations in the signalling
pathways, which often generate undesired pleiotropic effects, the
modifications in the regulation of transcription factors which are
locally expressed, such as BRC1, which exclusively acts in the axillary
buds, could be more easily tolerated. The transcriptional regulations
have played a key role in the evolution of many morphological features.
Indeed, during the domestication of corn, the genetic improvement for
obtaining plants with a strong apical dominance, gave rise to the
selection of plants that overexpress Tb1. CYCLOIDEA, another
transcription factor of the TCP family, has been responsible for the
evolution of floral bilateral symmetry, a morphological innovation which
has evolved independently in different clades.

[0005] The control of the development of the axillary buds has a great
applied potential since knowing its genetic bases allows us to control
the architecture of plants of agronomic interest.

[0006] By inhibition of the axillary development we can promote the growth
in a single axis favouring long stems and with few nodes as is desirable,
for example, in species of ligneous plants which are used for wood
production, others that are grown at high density such as gramineae or
those wherein the side stems are an obstacle for mechanized collection.
We can favour the contribution of nutrients to the axes which are
developing fruits (e.g. tomato) or extending the storage life of certain
products whose shoots reduce their quality (e.g. potatoes, onions,
garlic). The classic improvement has made it possible to obtain varieties
with a single stem or "monostem" in some species (e.g. sunflower);
however, in others (e.g. tobacco, tomato) it has not been possible to
achieve having this character in high production lines. The alternative
techniques used for obtaining plants with a single stem (manual
elimination of side branches, application of chemical products) not only
make the production more expensive, but they favour the propagation of
diseases and may entail problems of environmental pollution. Favouring
axillary development, we can generate shrubby architectures and increase
the production of leaves and flowers, elements appreciated in ornamental
species or in those where the fruit are the products of consumption. The
increase in the formation of shoots also has interest in species which
are used for the carpeting of land, wherein compact growth is valued
(e.g. gramineae for lawns or pastures). It would be of great ecological
value to promote the intercalated growth in creeper species adapted to
arid lands threatened by erosion wherein grass is costly to maintain. The
production of new shoots also has importance in plant propagation and in
vitro culture.

[0007] Finally, in certain ligneous species, the control of sprouting of
the axillary buds whose physiological and hormonal regulation is
comparable with that of herbaceous plants has great economic importance.
In vines, cherry trees, apple trees and ligneous species, the axillary
buds require an exposure to the cold during days or weeks to sprout.
These species have been begun to be grown in warm countries (e.g. Brazil
and Thailand) wherein low temperatures are not usually reached, for which
reason the farmers are obliged to use, to make the buds sprout, very
toxic chemical treatments (hydrocyanic acid, dinitro-orthocresol), or
costly hormonal treatments which are quickly degraded and produce
undesired effects.

[0008] The Solanaceae, and among them the tomato plant (Solanum
lycopersicum) and the potato plant (Solanum tuberosum), are plants of
great economic importance, where some of their agricultural
characteristics of interest depend on the activity of their axillary
buds. The sprouting of the buds alters the relation between the
production and consumption of photoassimilates, and can affect
production.

[0009] Therefore in fields such as agriculture, forestry and horticulture,
it would be of great interest to be able to control the development of
the axillary buds and the elongation of branches.

DESCRIPTION OF THE INVENTION

[0010] The authors of the present invention have isolated and researched
the role of the orthologous genes of Teosinte branched1 of corn and
BRANCHED1 (BRC1) of Arabidopsis in two species of the Solanaceae family,
the tomato plant (Solanum lycopersicum L.) and the potato plant (Solanum
tuberosum L.). These genes code for transcription factors of the TCP
family. The TCP proteins, exclusive of plants, are transcription factors
with a BHLH domain which gives DNA linking capacity and to other
proteins. In Arabidopsis the role of BRC1 has been demonstrated as
repressor during the initiation of the axillary meristems, the
development of the buds and branch growth.

[0011] The authors have found that there are two genes related to BRC1 in
each species (called SIBRC1L1 and SIBRC1L2, in the tomato plant and
StBRC1L1 and StBRC1L2 in the potato plant). They have also demonstrated
that, in both species, BRC1L1 and BRC1L2 play a fundamental role in
suppressing the development of axillary buds and branch elongation. In
the potato, StBRC1L1 and StBRC1L2 also control the formation of the
stolons and their branching, and the sprouting of the tuber eyes. BRC1L1
and BRC1L2 are specifically expressed in axillary buds but their
expression levels are different for each gene. The function loss
phenotype of each one indicates that, although both control the
branching, each gene has a certain degree of specialization and
functional divergence: in the potato, StBRC1L1 would preferably control
the branching of the stolons and StBRC1L2 the elongation of aerial
branches; in the tomato plant, SIBRC1L2 could play a more important role
than SIBRC1L1 in the control of the branch elongation.

[0012] Therefore, the sequences of nucleic acids that code for the
proteins product of these genes, promoters and the genetic constructions
product of this invention constitute a valuable tool for manipulation of
the development of the axillary buds, and the branching control, to
increase plant yield, and in particular of the potato and the tomato. The
invention also relates to the genetic constructions comprising these
sequences, as well as transformed cells, vectors and transgenic plants
which incorporate them. It also relates to agents modulating expression,
and therefore, biological activity, of these genes, as well as new
compositions including these modulating agents, and the use of these
sequences, genetic constructions, modulating agents and compositions for
the manipulation of the axillary buds, the growth and the branching of
the plants, and, in particular, of the tomato plant and of potato.

[0013] The present invention also comprises methods for manipulating the
plant architecture, in particular the branching, and therefore the yield
of said plants incorporating the expression and/or inhibition
constructions of the invention.

[0014] In the particular case of these two species of Solanaceae, the
inhibition of the expression of the new genes (SIBRC1L1, SIBRC1L2,
StBRC1L1, StBRC1L2) by RNA interfering technology (RNAi), increases
aerial branching in the case of the tomato plant (only the inhibition of
SIBRC1L2) and, in the case of the potato plant further increases the
production of stolons and their branching, increasing the agricultural
yield of this species. Increasing the expression of these new genes, in
contrast, would give rise to a reduction in the number of branches, in
the case of the tomato plant, favouring the contribution of nutrients to
the axes which are developing fruit, and avoiding the use of alternative
techniques used for obtaining plants with a single stem (such as the
manual pruning of the side branches or the application of chemical
products) which not only make production more expensive, but also favour
the propagation of disease and may entail problems of environmental
contamination.

[0015] Therefore, a first aspect of this invention relates to an isolated
RNA or DNA polynucleotide, hereinafter first polynucleotide of the
invention, capable of being translated into an amino acid sequence
comprising a peptide having an identity with SEQ ID NO: 1, selected from
any of the following:

[0016] a) at least 95%, or

[0017] b) at least 99%.

[0018] In a preferred embodiment of this aspect of the invention, the
isolated RNA or DNA polynucleotide is capable of being translated into
amino acid sequence SEQ ID NO: 1.

[0019] Another aspect of this invention relates to an isolated RNA or DNA
polynucleotide, hereinafter second polynucleotide of the invention,
capable of being translated into an amino acid sequence comprising a
peptide having an identity with SEQ ID NO: 2 selected from any of the
following:

[0020] a) at least 95%, or

[0021] b) at least 99%.

[0022] In a preferred embodiment of this aspect of the invention, the
isolated RNA or DNA polynucleotide is capable of being translated into
amino acid sequence SEQ ID NO: 2.

[0023] Another aspect of this invention relates to an isolated RNA or DNA
polynucleotide, hereinafter third polynucleotide of the invention,
capable of being translated into an amino acid sequence comprising a
peptide having an identity with SEQ ID NO: 3 selected from any of the
following:

[0024] a) at least 95%, or

[0025] b) at least 99%.

[0026] In a preferred embodiment of this aspect of the invention, the
isolated RNA or DNA polynucleotide is capable of being translated into
amino acid sequence SEQ ID NO: 3.

[0027] Another aspect of this invention relates to an isolated RNA or DNA
polynucleotide, hereinafter fourth polynucleotide of the invention,
capable of being translated into an amino acid sequence comprising a
peptide having an identity with SEQ ID NO: 4 selected from any of the
following:

[0028] a) at least 95%, or

[0029] b) at least 99%.

[0030] In a preferred embodiment of this aspect of the invention, the
isolated RNA or DNA polynucleotide is capable of being translated into
amino acid sequence SEQ ID NO: 4.

[0031] The gene SIBRC1L1 is translated into two proteins, a long one, of
346 amino acids (SEQ ID NO: 1) and another short one, of 325 amino acids
(SEQ ID NO: 50). Therefore, another aspect of this invention relates to
an isolated RNA or DNA polynucleotide, hereinafter eleventh
polynucleotide of the invention, capable of being translated into an
amino acid sequence comprising a peptide having an identity with SEQ ID
NO: 50 selected from any of the following:

[0032] a) at least 95%, or

[0033] b) at least 99%.

[0034] In a preferred embodiment of this aspect of the invention, the
isolated RNA or DNA polynucleotide is capable of being translated into
amino acid sequence SEQ ID NO: 50.

[0035] The authors of the present invention have also detected the
regulatory expression sequences of said genes, which are capable of
directing the expression of a gene of interest in axillary meristems but
not in apical meristems in the tomato. The use of a promoter such as that
provided by this invention makes it possible to genetically manipulate
the plants and obtain plants with improved characteristics, making it
possible to modify the plant architecture altering the growth or
development of its axillary buds without altering the growth of the main
axis.

[0036] Therefore, another aspect of this invention relates to an isolated
RNA or DNA polynucleotide, hereinafter fifth polynucleotide of the
invention, capable of directing the expression of a gene of interest in
the axillary buds, having an identity with SEQ ID NO: 5 selected from any
of the following:

[0037] a) at least 95%, or

[0038] b) at least 99%.

[0039] In a preferred embodiment of this aspect of the invention, the
isolated RNA or DNA polynucleotide has the nucleotide sequence included
in SEQ ID NO: 5.

[0040] Another aspect of this invention relates to an isolated RNA or DNA
polynucleotide, hereinafter sixth polynucleotide of the invention,
capable of directing the expression of a gene of interest in the axillary
buds, having an identity with SEQ ID NO: 6 selected from any of the
following:

[0041] a) at least 95%, or

[0042] b) at least 99%.

[0043] In a preferred embodiment of this aspect of the invention, the
isolated RNA or DNA polynucleotide has the nucleotide sequence included
in SEQ ID NO: 6.

[0044] It can be expected that the degree of identity/similarity of the
proteins homologous to those included in sequences SEQ ID NO: 1 and SEQ
ID NO: 2 (for the tomato plant), and SEQ ID NO: 3, SEQ ID NO: 4 (for the
potato), are, in different varieties and subspecies of Solanum
lycopersicum L. and Solanum tuberosum L., of at least 80% or greater, and
more preferably of at least 85%, 90, 95% or 99%. The correspondence
between the amino acid sequence(s) of the putative sequence(s) and the
sequences included in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID
NO: 4 can be determined by methods known in the state of the art. The
methods for sequence comparison are known in the state of the art, and
include, although without being limited to them, the program BLASTP or
BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215: 403-410 (1999).

[0045] The term "homology", as used in this specification, refers to the
similarity between two structures due to a common evolutionary ancestry,
and more specifically, to the similarity between two or more sequences of
nucleotides or amino acids. Since two sequences are considered homologous
if they have the same evolutionary origin, in general, it is assumed that
values of similarity or identity higher than 95% would indicate homology.
We can consider, therefore, that percentages of identity of, at least,
99%, could maintain the function of the orthologous amino acid sequences
included in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0046] The term "orthologous" refers to homologous structures of different
species, having a common ancestor, and in particular, to the similarity
between two or more sequences of nucleotides or amino acids.

[0047] The term "identity", as used in this specification, refers to the
proportion of identical nucleotides or amino acids between two nucleotide
or amino acid sequences compared. The methods of comparison of sequences
are known in the state of the art, and include, but are not limited to,
the GAG program, including GAP (Devereux et al., Nucleic Acids Research
12: 287 (1984) Genetics Computer Group University of Wisconsin, Madison,
(WI); BLAST, BLASTP or BLASTN, and FASTA (Altschul et al., J. Mol. Biol.
215: 403-410 (1999).

[0048] In another aspect of the invention a genetic construction of DNA or
RNA is provided, hereinafter first genetic construction of the invention,
comprising one of the following types of sequences:

[0049] a) sequence of nucleotides, comprising, at least, the first
polynucleotide of the invention, or the coding sequence of SEQ ID NO: 1,
for its transcription in vitro or in vivo, or

[0050] b) sequence of nucleotides, corresponding to a gene expression
system or vector comprising the first polynucleotide of the invention,
operatively linked to, at least, one promoter which directs the
transcription of said sequence of nucleotides, and to other sequences
necessary or appropriate for the transcription and their suitable
regulation in time and place, for example, initiation and termination
signals, cleavage sites, polyadenylation signals, replication origin,
transcriptional enhancers, transcriptional silencers, etc.

[0051] In a preferred embodiment of this aspect of the invention, the
promoter is the fifth polynucleotide of the invention.

[0052] In another aspect of the invention a genetic construction of DNA or
RNA is provided hereinafter second genetic construction of the invention,
comprising one of the following types of sequences:

[0053] a) sequence of nucleotides, comprising, at least, the second
polynucleotide of the invention, or the coding sequence of SEQ ID NO: 2,
for its transcription in vitro or in vivo, or

[0054] b) sequence of nucleotides, corresponding to a gene expression
system or vector comprising the second polynucleotide of the invention,
operatively linked to, at least, one promoter which directs the
transcription of said sequence of nucleotides, and to other sequences
necessary or appropriate for the transcription and their suitable
regulation in time and place, for example, initiation and termination
signals, cleavage sites, polyadenylation signals, replication origin,
transcriptional enhancers, transcriptional silencers, etc.

[0055] In a preferred embodiment of this aspect of the invention, the
promoter is the sixth polynucleotide of the invention.

[0056] In another aspect of the invention a genetic construction of DNA or
RNA is provided, hereinafter third genetic construction of the invention,
comprising one of the following types of sequences:

[0057] a) sequence of nucleotides, comprising, at least, the third
polynucleotide of the invention, or the coding sequence of SEQ ID NO: 3,
for its transcription in vitro or in vivo, or

[0058] b) sequence of nucleotides, corresponding to a gene expression
system or vector comprising the third polynucleotide of the invention,
operatively linked to, at least, one promoter which directs the
transcription of said sequence of nucleotides, and to other sequences
necessary or appropriate for the transcription and their suitable
regulation in time and place, for example, initiation and termination
signals, cleavage sites, polyadenylation signals, replication origin,
transcriptional enhancers, transcriptional silencers, etc.

[0059] In another aspect of the invention a genetic construction of DNA or
RNA is provided, hereinafter fourth genetic construction of the
invention, comprising one of the following types of sequences:

[0060] a) sequence of nucleotides, comprising, at least, the fourth
polynucleotide of the invention, or the coding sequence of SEQ ID NO: 4,
for its transcription in vitro or in vivo, or

[0061] b) sequence of nucleotides, corresponding to a gene expression
system or vector comprising the fourth polynucleotide of the invention,
operatively linked to, at least, one promoter which directs the
transcription of said sequence of nucleotides, and to other sequences
necessary or appropriate for the transcription and their suitable
regulation in time and place, for example, initiation and termination
signals, cleavage sites, polyadenylation signals, replication origin,
transcriptional enhancers, transcriptional silencers, etc.

[0062] In another aspect of the invention a genetic construction of DNA or
RNA is provided, hereinafter fifth genetic construction of the invention,
comprising one of the following types of sequences:

[0063] a) sequence of nucleotides, comprising, at least, the eleventh
polynucleotide of the invention, or the coding sequence of SEQ ID NO: 50,
for its transcription in vitro or in vivo, or

[0064] b) sequence of nucleotides, corresponding to a gene expression
system or vector comprising the eleventh polynucleotide of the invention,
operatively linked to, at least, one promoter which directs the
transcription of said sequence of nucleotides, and to other sequences
necessary or appropriate for the transcription and their suitable
regulation in time and place, for example, initiation and termination
signals, cleavage sites, polyadenylation signals, replication origin,
transcriptional enhancers, transcriptional silencers, etc.

[0065] In another aspect of the invention a genetic construction of DNA or
RNA is provided, hereinafter sixth genetic construction of the invention,
comprising one of the following types of sequences:

[0066] a) sequence of nucleotides, comprising the fifth polynucleotide of
the invention, or

[0067] b) sequence of nucleotides, comprising the sixth polynucleotide of
the invention, operatively linked to a gene of interest. Said
construction enables directing the expression of the gene of interest
specifically in axillary buds.

[0068] A great number of these constructions, systems or expression
vectors may be obtained by conventional methods known by persons skilled
in the art and form part of the present invention.

[0069] A "vector" is a replicon, or an integrative vector, whereto another
polynucleotide segment has been linked, to perform the replication and/or
expression of the linked segment.

[0070] A "replicon" is any genetic element which behaves as an autonomous
unit of polynucleotide replication within a cell; i.e. capable of
replicating under its own control.

[0071] An integrative vector is any genetic element which is integrated
and maintains stable in the cell genome.

[0072] "Control sequence" relates to polynucleotide sequences necessary to
carry out the expression of the sequences whereto they are linked. The
nature of said control sequences differs depending on the host organism;
in prokaryotes, said control sequences generally include a promoter, a
ribosomal binding site and termination signals; in eukaryotes, generally,
said control sequences include promoters, termination signals,
intensifiers and, on occasions, silencers. It is aimed that the term
"control sequences" includes, at minimum, all the components whose
presence is necessary for expression and it can also include additional
components whose presence is advantageous.

[0073] As used here, the term "promoter" refers to a region of the DNA
upstream from the start point of the transcription, and particular
therein, which is capable of initiating the transcription in a plant
cell, whether the origin of the promoter is a plant or not. Examples of
promoters include, but are not limited to, promoters obtained from
plants, plant virus, and bacteria that may express genes in plant cells,
such as Agrobacterium or Rhizobium. Examples of promoters under the
control of development include promoters that preferably initiate
transcription in certain tissues, such as leaves, roots or seeds. Said
promoters are denominated in this specification as preferable of a type
of tissue. There are other promoters which initiate transcription in a
certain type of tissues, and are called "specific tissues". An
"inducible" or "repressible" promoter is a promoter which is under the
control of the environment. Examples of environmental conditions that may
affect transcription are anaerobic conditions, or the presence of light.
The promoters of specific tissue, preferred tissue, specific of a cell
type or inducible promoters are types that constitute the class of
"non-constitutive" promoters the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter which is active in the majority of
environmental conditions.

[0074] "Operatively linked" relates to a juxtaposition wherein the
components thus described have a relation that allows them to operate in
the intended manner. A control sequence "operatively linked" to a
sequence which transcribes the nucleotide sequence of the invention is
linked so that the expression of the encoding sequence is achieved in
conditions with the control sequences.

[0075] An "encoding sequence" or "coding sequence" is a sequence of
polynucleotides which is transcribed to mRNA and/or is translated into a
polypeptide when it is under the control of suitable regulating
sequences. The limits of the coding sequence are determined by a
translation initiation codon at end 5' and a translation termination
codon at end 3'. A coding sequence may include, but is not limited to,
mRNA, cDNA, and recombinant polynucleotide sequences.

[0076] The terms "polynucleotide" and "nucleic acid" are used here
interchangeably, referring to polymeric forms of any length, both
ribonucleotides (RNA) and deoxyribonucleotide (DNA).

[0077] The terms "amino acid sequence", "peptide", "oligopeptide",
"polypeptide" and "protein" are used here interchangeably, and refer to a
polymeric form of amino acids of any length which may or may not be
chemically or biochemically modified.

[0078] Another aspect of the invention relates to the use of the
polynucleotides of the invention, or the genetic constructions of the
invention, in the production of cells and transgenic plants which have a
modified plant architecture.

[0079] In this specification "plant architecture" is understood as the sum
of the observable structural properties of an organism (plant), for
example, the trend of the plants to grow vertically or shrub-like,
together with the functional properties that constitute the phenotype of
said organism, which is the result of the interaction between the
genotype and the environment.

[0080] "Plant" in this specification is understood to be all organisms
that can be classified within the kingdom Viridiplantae, including green
algae and land plants (Embryophyta).

[0081] The organisms of the genus Solanum belong to the Superkingdom
Eukaryota, Kingdom Viridiplantae, Phylum Streptophyta, Subclass
Asteridae, Orden Solanales, Family Solanaceae. Solanum tuberosum is the
scientific name of the potato plant, and Solanum lycopersicum that of the
tomato plant.

[0082] In another aspect of the invention a method is provided to modify
the plant architecture of a plant, comprising:

[0083] a) transfecting the polynucleotides or the genetic constructions of
the invention in a cell or culture of host plant cells,

[0084] b) growing the cell or the culture of host plant cells in a
suitable medium, until regenerating a complete plant.

[0085] A "host" or "host cell" as used in this specification relates to an
organism, cell or tissue, particularly to a plant cell, which serves as
target or recipient of the transfected elements (for example, the
polynucleotides or the genetic constructions of the invention). A host
cell may also indicate a cell or host that expresses a recombinant
protein of interest (for example, the product of the expression of the
polynucleotides of the invention) where the host cell is transformed with
an expression vector containing the polynucleotides of the invention or
also the promoters of the invention which direct the expression of a gene
of interest.

[0086] "Transfecting" or "transgenesis" in this specification is
understood as the process of transferring foreign DNA to an organism,
which becomes in this way known as "transgenic".

[0087] The term "transgenic" is used in the context of the present
invention to describe plants wherein a foreign sequence of DNA has been
incorporated stably, and in particular the polynucleotides or the genetic
constructions of the invention.

[0088] In a preferred embodiment of this aspect of the invention, the
cell, the culture of plant cells and/or the plant may be taxonomically
classified in the species Solanum tuberosum L. In a preferred embodiment
of this aspect of the invention, the cell, the culture of plant cells
and/or the plant may be taxonomically classified in the species Solanum
lycopersicum .

[0089] The method to modify the plant architecture of a plant provided by
the invention comprises any process of plant transformation wherein the
allogenous elements introduced comprise the polynucleotides of the
invention or the genetic constructions of the invention.

[0090] In another aspect of the invention a method is provided to express
a gene of interest in the axillary meristems of a plant comprising:

[0091] a) transfecting the polynucleotides or the genetic constructions of
the invention in a cell or culture of host plant cells,

[0092] b) growing the cell or the culture of host plant cells in a
suitable medium, until regenerating a complete plant.

[0093] In a preferred embodiment of this aspect of the invention, the
cell, the culture of plant cells and/or the plant may be taxonomically
classified in the species Solanum tuberosum L. In a preferred embodiment
of this aspect of the invention, the cell, the culture of plant cells
and/or the plant may be taxonomically classified in the species Solanum
lycopersicum.

[0094] The method to express a gene of interest in the axillary meristems
of a plant provided by the invention comprises any process of plant
transformation wherein the allogenic elements introduced comprise the
polynucleotides of the invention or the genetic constructions of the
invention.

[0095] The polynucleotides and some of the genetic constructions of the
present invention are expressed in temporally and spatially regulated
form (for example, in certain stages of development and in certain
tissues, axillary buds) and at controlled levels. An aspect of the
present invention consists of altering (increasing or decreasing) said
expression levels.

[0096] The present invention also comprises modulating agents of the
expression of the proteins coded by the polynucleotides of the invention,
and/or of the genes constituting coding for these proteins in the tomato
plant and the potato (SIBRC1L1, SIBRC1L2, StBRC1L1 and StBRC1L2). With
the development of anti-sense technology, sequences of specific
nucleotides complementary to a certain sequence of DNA or RNA, could form
complexes and block the transcription or translation. Furthermore, with
the progress of post-transcriptional gene silencing and, in particular,
interfering RNA (or RNAi), tools have been developed which allow the
specific inhibition of the expression of a gene. The inhibition of the
expression of the genes SIBRC1L1, SIBRC1L2, StBRCIU and StBRC1L2 would
hence constitute the inhibition of its biological activity, allowing the
modulation of said activity in the plant.

[0097] In the context of the present invention, SIBRC1L1 is defined by a
sequence of nucleotides or polynucleotide, which constitutes the coding
sequence of the protein SIBRC1L1, and would comprise different variants
of:

[0100] c) nucleic acid molecules the sequence whereof differs from a)
and/or b) due to the degeneration of the genetic code,

[0101] d) nucleic acid molecules which code for a polypeptide comprising
the amino acid sequence with an identity of at least 95%, 98% or 99% with
the SEQ ID NO: 1 or with the SEQ ID NO: 50, wherein the polypeptide coded
by said nucleic acids have the activity and the structural
characteristics of the protein SIBRC1L1.

[0102] A nucleotide sequence capable of being translated into SEQ ID NO: 1
could be, but without being limited to, the sequence included in SEQ ID
NO: 7.

[0103] In the context of the present invention, SIBRC1L2 is defined by a
sequence of nucleotides or polynucleotide, which constitutes the coding
sequence of the protein SIBRC1L2, and would comprise different variants
from:

[0106] c) nucleic acid molecules the sequence whereof differs from a)
and/or b) due to the degeneration of the genetic code,

[0107] d) nucleic acid molecules which code for a polypeptide comprising
the amino acid sequence with an identity of at least 95%, 98% or 99% with
the SEQ ID NO: 2, wherein the polypeptide coded by said nucleic acids
have the activity and the structural characteristics of the protein
SIBRC1L2.

[0108] A nucleotide sequence capable of being translated into SEQ ID NO: 2
could be, but without being limited to, the sequence included in SEQ ID
NO: 8.

[0109] In the context of the present invention, StBRC1L1 is defined by a
sequence of nucleotides or polynucleotide, which constitutes the coding
sequence of the protein StBRC1L1, and would comprise different variants
of:

[0112] c) nucleic acid molecules the sequence whereof differs from a)
and/or b) due to the degeneration of the genetic code,

[0113] d) nucleic acid molecules which code for a polypeptide comprising
the amino acid sequence with an identity of at least 95%, 98% or 99% with
the SEQ ID NO: 3, wherein the polypeptide coded by said nucleic acids
have the activity and the structural characteristics of the protein
StBRC1L1. A nucleotide sequence capable of being translated into SEQ ID
NO: 3 could be, but without being limited to, the sequence included in
SEQ ID NO: 9.

[0114] In the context of the present invention, StBRC1L2 is defined by a
sequence of nucleotides or polynucleotide, which constitutes the coding
sequence of the protein StBRC1L2, and would comprise different variants
of:

[0117] c) nucleic acid molecules the sequence whereof differs from a)
and/or b) due to the degeneration of the genetic code,

[0118] d) nucleic acid molecules which code for a polypeptide comprising
the amino acid sequence with an identity of at least 95%, 98% or 99% with
the SEQ ID NO: 4, wherein the polypeptide coded by said nucleic acids
have the activity and the structural characteristics of the protein
StBRC1 L2.

[0119] A nucleotide sequence capable of being translated into SEQ ID NO: 4
could be, but without being limited to, the sequence included in SEQ ID
NO: 10.

[0120] Furthermore, due to the existence of different alleles, the amino
acid sequence whereinto the gene StBRC1L2 is translated may vary, being
included in an alternative sequence in SEQ ID NO: 51. A nucleotide
sequence capable of being translated into SEQ ID NO: 51 could be, but
without being limited to, the sequence included in SEQ ID NO: 52.

[0121] "Antisense polynucleotides" are understood to be chains of
ribonucleotides or deoxyribonucleotides which may inhibit the activity of
these genes by one of these two mechanisms:

[0122] 1--Interfering the transcription, on hybridizing with the
structural gene or in a regulator or promoter region of the gene which
codes for these transcription factors (SIBRC1L1, SIBRC1L2, StBRC1L1 and
StBRC1L2). Since the transcription or expression is effectively blocked
by the hybridization of the antisense oligonucleotide with the DNA, the
production of these transcription factors decreases.

[0123] 2--The linking of the antisense oligonucleotide in the cytoplasm
with the mRNA, interfering with the formation of the actual translation
complex, inhibiting complex of translation, inhibiting the translation of
the mRtoA into protein.

[0124] The post transcriptional gene silencing, and in particular of the
interfering RNA also give rise to less production of these transcription
factors. The interfering RNA or interfering RNA (or iRNA), is a molecule
of RNA which causes the degradation of the RNA of specific genes. In this
specification, the interfering RNA includes both the siRNA (small
interfering RNA, and the tsRNA ("trans-splicing RNA"), VIGS ("Virus
induced gene silencing") and the miRNA or microARN. The siRNA are double
strands of RNA, perfectly complementary, of approximately 20-21
nucleotides (nt) with 2 free nucleotides at each end 3'.

[0125] Each strand of RNA has a phosphate 5' group and a hydroxyl (--OH)
3' group. This structure comes from the processing carried out by Dicer,
an enzyme which cuts long strands of double strand (dsRNA) in siRNAs. One
of the strands of the siRNA (the antisense) is assembled in a protein
complex called RISC (RNA-induced silencing complex), which uses the
strand of siRNA as guide to identify the complementary messenger RNA. The
RISC complex catalyzes the cleavage of the complementary mRNA in two
halves, which are degraded by the cellular machinery, thus blocking the
gene expression. The miRNAs are small interfering RNAs which are
generated from specific precursors coded in the genome, which on being
transcribed is folded in intramolecular hairpins which contain segments
of imperfect complementarity. The processing of the precursors generally
occurs in two stages, catalysed by two enzymes, Drosha in the nucleus and
Dicer in the cytoplasm. One of the strands of the miRNA (the antisense),
as occurs with the siRNAs, is incorporated in a complex similar to the
RISC. Depending on the degree of complementarity of the miRNA with the
mRNA, the miRNAs may either inhibit the translation of the mRNA or induce
their degradation. However, unlike the pathway of the siRNAs, the
degradation of mRNA mediated by miRNAs starts with the enzymatic
elimination of the poly-A tail of the mRNA.

[0126] Therefore, it could be any siRNA or miRNA capable of hybridizing a
nucleic acid molecule which codes these transcription factors (SIBRC1L1,
SIBRC1L2, StBRC1L1 and StBRC1L2), or an RNA construction which at least
contains any of the possible sequences of siRNA or miRNA nucleotides
capable of inhibiting the translation of the orthologous proteins of BRC1
of the invention, and without prejudice to additionally forming part of
the present invention any of the sequences and RNA constructions of the
invention mentioned above which are object of modifications, preferably
chemical, which lead to a greater stability against the action of
ribonuclease and with this a greater efficiency. Without said
modifications supposing the alteration of its mechanism of action, which
is the specific link to the RISC complex (RNA-induced silencing complex),
activating it and manifesting a helicase activity which separates the two
strands leaving only the antisense strand associated to the complex.

[0127] Additionally, it is evident for a person skilled in the art that a
great quantity of mRNA polynucleotides may be translated into proteins
SIBRC1L1, SIBRC1L2, StBRC1L1 and StBRC1L2 as a consequence, for example,
of the genetic code being degenerated. Any siRNA or miRNA capable of
inhibiting the translation of these mRNA also form part of the invention.

[0128] The authors of the present invention have developed four sequences
of interfering RNA, two of them aimed at reducing the mRNA levels of the
genes SIBRC1L1 and SIBRC1L2 of the tomato plant (seventh--SEQ ID NO:
11--and eighth--SEQ ID NO: 12--polynucleotide of the invention,
respectively) and two of them aimed at reducing the mRNA levels of the
genes StBRC1L1 and

[0129] StBRC1L2 of the potato (ninth and tenth polynucleotide of the
invention, SEQ ID NO: 13 and SEQ ID NO: 14 respectively). Therefore,
another aspect of the invention relates to a sequence which is selected
from the list comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or
SEQ ID NO: 14. The sequences of interfering RNA of the invention would
serve to modify the plant architectures of the plants, and in particular
of the tomato plant and of the potato. As demonstrated in the examples of
the present invention, the inhibition of the SIBRC1L1 gene of the tomato
plant does not produce an apparent modification (with respect to the
elongation of the aerial branches) of the plant architecture (or of its
phenotype). This is indicative that, although both genes control the
branching, each one has a certain degree of specialization and functional
divergence so that in the tomato plant, SIBRC1L2 could have a more
important than SIBRC1L1 in the control of branch elongation. On the other
hand, in the potato plant, StBRC1 L1 would preferably control the
branching of the stolons and StBRC1L2 the elongation of aerial branches.

[0130] A genetic construction of DNA also forms part of the present
invention, which would direct the in vitro or intracellular transcription
of the sequence of siRNA, miRNA, or RNA construction of the invention,
and comprising, at least, one of the following types of sequences: a)
sequence of DNA nucleotides, preferably double chain, comprising, at
least, the sequence of the siRNA or miRNA of the invention or of the RNA
construction of the invention for its transcription, or, b) sequence of
DNA nucleotides, preferably double chain, corresponding to a gene
expression system or vector comprising the sequence which transcribes to
the RNA sequence of the invention operatively linked to, at least, one
promoter which directs the transcription of said sequence of nucleotides
of interest, and to other sequences necessary or appropriate for the
transcription and their suitable regulation in time and place, for
example, initiation and termination signals, cleavage sites,
polyadenylation signals, replication origin, transcriptional enhancers,
transcriptional silencers, etc. Said genetic construction could be used
in the modification of the plant architecture. Many of these
constructions, systems or expression vectors can be obtained by
conventional methods known by persons skilled in the art (Sambrook et al.
2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
Laboratory Press, New York). Examples of these constructions would be,
but without being limited to, the binary DNA plasmids used for the
generation of the lines 35SCaMV:: SIBRC1L1 RNAi, 35SCaMV:: SIBRC1L2 RNAi,
35SCaMV:: StBRC1L1 RNAi and 35SCaMV:: StBRC1L2 RNAi, and which are
included in FIG. 7 of this specification.

[0131] The preparation of other siRNA or miRNA sequences of the invention
or of the RNA constructions of the invention would be evident for a
person skilled in the art, and could be carried out by chemical
synthesis, which also permits the incorporation of chemical modifications
both in the different nucleotides of the product and the incorporation of
other chemical compounds at any of the ends. On the other hand, the
synthesis could also be performed enzymatically using any of the
available RNA polymerases. The enzymatic synthesis also allows chemical
modifications of the products or inhibitor RNAs.

[0132] The design of the siRNA or miRNA nucleotide sequences of the
invention would also be evident for a person skilled in the art. Thus,
for the siRNA it could be performed by a random design wherein 19-25
bases of the target mRNA are selected without bearing in mind the
sequence or the positional information it has in the transcript. Another
non-limiting alternative of the present invention would be the
conventional design by simple parameters developed by the pioneers of the
technique (Calipel et al., 2003. J Biol. Chem. 278(43): 42409-12418)
completed with BLAST analysis of nucleotides. Another possibility could
be a rational design wherein a computer process is used aimed at
identifying the optimum targets of siRNA in a mRNA. The target sequences
are analysed in groups of 19 nucleotides at the same time and are
identified as those which have the best characteristics depending on an
algorithm which incorporates a great number of thermodynamic and sequence
parameters.

[0133] The antibodies capable of linking to proteins SIBRC1L1, SIBRC1L2,
StBRC1L1 and StBRC1L2 can be used to inhibit the activity of said
proteins, therefore modulating said activity. Therefore, in another
preferred embodiment of this aspect of the invention, the modulating
agent is selected from antibodies, fragments thereof, or any of their
combinations. The antibodies may be polyclonal (typically include
different antibodies directed against different determinants or epitopes)
or monoclonal (directed against a single determinant in the antigen. The
monoclocal antibody may be altered biochemically, by genetic
manipulation, or may be synthetic, lacking, possibly, the antibody in its
totality or in parts, of portions which are not necessary for the
recognition of the proteins SIBRC1L1, SIBRC1L2, StBRC1L1 and StBRC1L2 and
being substituted by others which communicate additional advantageous
properties to the antibody. The antibody may also be recombinant,
chimerical, synthetic or a combination of any of the previous.

[0134] The term "antibody" as used in this specification, relates to
molecules of immunoglobulins and immunological active portions of
immunoglobulin molecules, i.e. molecules that contain an antigen fixation
site which is specifically bound (immunoreactance) with the proteins
SIBRC1L1, SIBRC1L2, StBRC1L1 and StBRC1L2. Examples of portions of
immunologically active immunoglobulin molecules include fragments F(ab)
and F(ab')2 which may be generated by treating the antibody with an
enzyme such as pepsin. It may be a monoclonal or polyclonal antibody.

[0135] A "recombinant antibody or polypeptide" (rAB) is one which has been
produced in a host cell which has been transformed or transfected with
the coding nucleic acid of the polypeptide, or produces the polypeptide
as a result of homologous recombination.

[0136] These rAC can be expressed and directed towards specific cellular
subcompartments when the appropriate sequences for intracellular traffic
are incorporated. These antibodies are called intrabodies, and have
demonstrated their efficacy not only to deviate proteins from their
habitual compartment or block interactions between proteins involved in
signalling pathways, but also to activate intracellular proteins.

[0137] Part of the invention is also the genetic constructions of DNA
capable of transcribing to a peptide, antibody or fragment of antibody,
for their use in a modification of the plant architecture. Said genetic
construction of DNA would direct the in vitro or intracellular
transcription of the sequence of the antibody or fragment thereof, and
comprises, at least, one of the following types of sequences: a) sequence
of DNA nucleotides, preferably double chain, comprising, at least, the
coding sequence of the antibody of the invention or of the fragment of
antibody of the invention for its in vitro or intracellular
transcription, b) sequence of DNA nucleotides, preferably double chain,
corresponding to a gene expression system or vector comprising the coding
sequence of the sequence of antibody or fragment of antibody of the
invention operatively linked to, at least, one promoter which directs the
transcription of said sequence of nucleotides of interest, and to other
sequences necessary or appropriate for the transcription and their
suitable regulation in time and place, for example, initiation and
termination signals, cleavage sites, polyadenylation signals, replication
origin, transcriptional enhancers, transcriptional silencers, etc. for
their use in the modification of the plant architecture.

[0138] Ribozymes could also be used as modulating agents of the activity
of the proteins SIBRC1L1, SIBRC1L2, StBRC1L1 and StBRC1L2. A "ribozyme"
as understood in the present invention, relates to a catalytic
polynucleotide (typically RNA), which may be built to specifically
recognize, by hybridization, a mRNA and fragment it or eliminate its
expression. The ribozymes may be introduced in the cell as catalytic RNA
molecules or as genetic constructions which are expressed to catalytic
molecules of RNA.

[0139] The compositions comprising the antisense oligonucleotides
antisense (siRNA, miRNA or the RNA construction), the antibodies, or the
genetic constructions modulating the expression of the genes SIBRC1L1,
SIBRC1L2, StBRC1L1 and StBRC1L2 of the invention also form part of the
invention. The compositions of the present invention allow the
transfection of the siRNA, miRNA or the RNA construction of the invention
to the interior of a cell, in vivo or in vitro. The transfection could be
carried out, but without being limited to, direct transfection or vectors
that facilitate the access of the siRNA, miRNA or the RNA construction to
the interior of the cell. Thus, examples of these vectors are, without
being limited to, virus, non-viral binary plasmids of DNA, and molecular
conjugates. Thus, for example, the siRNA of the present invention, as
well as RNA or DNA precursors of these siRNA, miRNA or RNA constructions
can be conjugated with release peptides or other compounds to favour the
transport of these RNA to the interior of the cell.

[0140] Another aspect relates to a seed, hereinafter seed of the
invention, the genetic material whereof integrates the isolated
polynucleotides of the invention (including also the modulating agents,
such as for example, but without being limited to, those set down in SEQ
ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14) or the genetic
constructions of the invention. In a preferred embodiment, the seed of
the invention can be taxonomically classified as belonging to the species
Solanum tuberosum L. In a preferred embodiment, the seed of the invention
can be taxonomically classified as belonging to the species Solanum
lycopersicum .

[0141] Another aspect relates to a plant cell, hereinafter plant cell of
the invention, the genetic material whereof integrates the isolated
polynucleotides of the invention or the genetic constructions of the
invention. Preferably, the plant cells of the invention can be
taxonomically classified as belonging to the species Solanum tuberosum L.
In another preferred embodiment, it can be taxonomically classified as
belonging to the species Solanum lycopersicum .

[0142] Another aspect relates to a culture of plant cells, hereinafter
culture of plant cells of the invention, the genetic material whereof
integrates the isolated polynucleotides of the invention or the genetic
constructions of the invention. Preferably, the plant cells of the
culture of the invention may be taxonomically classified as belonging to
the species Solanum tuberosum L. In another preferred embodiment, they
may be taxonomically classified as belonging to the species Solanum
lycopersicum .

[0143] The term "culture of cells" in this specification, refers to a
culture of cells isolated therefrom or a different type of tissue, or a
collection of said cells organized in parts of a plant or in tissues
(tissue cultures). Types of cultures of this type are, for example,
cultures of protoplasts, calluses (groups of undifferentiated plant cells
capable of regenerating a complete plant) and plant cells which are
isolated from plants or parts of the plants, such as embryos,
protoplasts, meristematic cells, pollen, leaves or anthers.

[0144] Another aspect of the invention relates to a group of cells, which
may be taxonomically classified as belonging to the species Solanum
tuberosum L the genetic material whereof integrates the isolated
polynucleotides of the invention or the genetic constructions of the
invention, and which form the tubers, the minitubers or the microtubers.

[0145] "Minitubers", or "papa seed" are known as small tubers of no more
than 3 cm diameter used to perform the large commercial plantations of
potato crops. Failing this, medium-sized tubers are used or parts of them
which have at least one eye (i.e. a bud).

[0146] Another aspect relates to a plant, hereinafter plant of the
invention, comprising the cells or the culture of plant cells of the
invention, and/or which has been obtained after the growth of the seed of
the invention. Said plant would integrate in its genetic material the
polynucleotides of the invention, and/or the genetic constructions of the
invention. Preferably, the plant cells of the culture of the invention
may be taxonomically classified as belonging to the species Solanum
tuberosum L. In another preferred embodiment, they may be taxonomically
classified as belonging to the species Solanum lycopersicum .

[0147] Modifications in the genes SIBRC1L1, SIBRC1L2, StBRC1L1 and
StBRC1L2, would therefore allow modifying the plant architecture of a
plant. Since in this invention it includes the sequence of these genes,
as well as the respective proteins into which they are translated, the
obtainment of plants whose plant architecture is modified could be done
by several methods.

[0148] By selection of spontaneous mutants: it must bear in mind that in
each cellular division there is a small probability that a genetic change
occurs, for which reason it is not surprising that in a great cell mass
the population is heterogeneous. This distribution may have problems of
yield since, in general, the variants have less production levels than
the parent population. These definitive changes (mutations) must be
distinguished from the phenotypical variations that depend on the
environmental conditions and which take place in the population that
expresses the same physiological modification, within the variations
permitted by its genotype. In spontaneous mutations, if the responsible
element of the mutation is not known, it is very difficult to
differentiate these phenotypical variations from those which have
modifications in the genes responsible for the plant architecture and
which are stables and hereditary. The present invention provides the
necessary tools to carry out a selection of those mutants not only by the
observation of the morphological characteristics of interest, but also by
the detection of mutations in the genes responsible for said mutations
(SIBRC1L1, SIBRC1L2, StBRC1L1 and StBRC1L2), designing a simple selective
screening process for a particular type of mutants. For example, those
plants could be morphologically selected, preferably the tomato plant or
the potato, which have an advantageous plant architecture, and to later
check if the genes SIBRC1L1, SIBRC1L2, StBRC1L1 and StBRC1L2 have
mutations with respect to a control wild genotype. Therefore, another
aspect of the invention relates to a tomato plant, a fruit, seed, cells,
group of cells or parts of the plant, which have a plant architecture
modified with respect to the control-type tomato plants, where the
modification of the plant architecture is due to non-transgenic mutations
in the genes SIBRC1L1 and SIBRC1L2 of tomato.

[0149] Another aspect of the invention relates to a potato plant, a fruit,
seed, cells, group of cells or parts of the plant, which have a plant
architecture modified with respect to the control-type potato plants,
where the modification of the plant architecture is due to non-transgenic
mutations in the genes StBRC1L1 and StBRC1L2 of the potato plant.

[0150] The term "genotype", as used in this specification, refers to the
hereditary or genetic constitution of an individual; all the genetic
material contained in a cell, whereto, in general, is called nuclear
material.

[0151] The term "phenotype", as used in this specification, relates to the
sum total of the observable structural and functional properties of an
organism product of the interaction between the genotype and the
environment.

[0152] The term "type" refers to the plant designated as the type of a
genus, subgenus, species, variety or another taxonomic category, the
"type" being, from a taxonomic standpoint, the simple element of a taxon
whereto the name is permanently assigned and whereon are based the
descriptive characteristics which meet the conditions of availability or
of valid publication. This specification also describes a tomato or
potato control plant with which other plants of the same taxonomic
category are compared to observe if its plant architecture has been
modified, to later analyse if the genes SIBRC1L1 and SIBRC1L2 (in the
tomato plant) and StBRC1L1 and StBRC1L2 (in the potato) have mutations
with respect to the genes of the control plant. In this way, it is
possible to distinguish the modifications in the plant architecture which
are due to physiological, environmental or another type of factors,
against those caused by mutations in the genes SIBRC1L1 and SIBRC1L2 (in
the tomato plant) and StBRC1 L1 and StBRC1L2.

[0153] The induced mutation process involves two stages, the treatment of
the population with the chosen mutagen and then the isolation of the
mutants for their later testing and selection. Inducing mutations in a
plant is a very valuable tool for the improvement of plants, especially
when it is desired to improve one or two easily identifiable
characteristics in a well-adapted species or variety. Furthermore, it has
the advantage that the variability caused by the induced mutations is not
essentially different from that caused by spontaneous mutations during
the evolution. The choice of a mutagenic agent depends in general on
practical considerations. In some of the cases it is more convenient to
use more than one instead of the mass use of just one. Until where the
isolation of the mutant is possible the improved character thereof must
be used (the plant architecture of interest) as selection factor. The
mutagenic agents may be grouped in physical (ultraviolet light, x-rays,
gamma rays, beta radiation, rapid neutrons, heavy ion beams) and
chemical. Most of the chemical mutagens belong to the group of the
alkylation agents (ethyl methanesulfonate (EMS), diethyl sulfate (dES), .
. . ) but there are other groups, such as analogues of bases (such as
5-bromouracil and 2-aminopurine) and structural mutagens (such as
proflavin or acridine orange).

[0155] Thus, for example, the seeds are subjected to the action of
chemical mutagens, which give rise to a series of mutagens in the genome
of said seeds. Said seeds grow giving rise to adult plants (M1), which
self-pollinate giving rise to generation M2. The DNA of the M2 plants is
subjected to a screening to see if it has mutations in the gene of
interest. Once the mutation has been identified in the gene of interest,
the seeds of the M2 plants carrying said mutation grow, giving rise to M3
plants, which are subjected to a screening to see it they manifest the
phenotypical characteristics associated with the gene of interest.

[0156] A person skilled in the art understands that a variety of plant
material may be subject to the mutagenesis process, including, but
without being limited to, seeds, pollen, cells or tissues of the plant.
The type of plant material which is subjected to mutagenesis modifies the
stage in the DNA of the plants is subjected to screening to find the
mutation. Thus, for example, when the pollen is subjected to mutagenesis
before the pollination of a plant, the resulting seeds give rise to M1
plants. Each cell of said M1 plants may contain the mutations induced in
the pollen, for which reason it is necessary to wait for the M2
generation to perform the screening. This process is known in the state
of the art as tilling.

[0157] Thus, another aspect of the invention relates to a method for
obtaining tomato plants with modified plant architecture, in comparison
with the wild control plant, comprising:

[0158] a) obtaining plant material from a tomato plant (parent),

[0159] b) subjecting the plant material of step (a) to a mutagenesis
process

[0160] c) culturing the mutated plant material until regenerating a
complete plant, and its descendants,

[0161] d) analysing the descendants of the plants of step (c) to detect at
least one mutation in at least one copy the orthologous genes of BRC1
(genes SIBRC1L1 and SIBRC1L2),

[0162] e) selecting the descendants with at least one mutation in at least
one copy of the genes SIBRC1L1 and SIBRC1L2 which have their plant
architecture modified in comparison with a control type plant,

[0163] f) optionally, culturing the plant selected to obtain descendants
which have said modification of the plant architecture.

[0164] In a preferred embodiment of this aspect of the invention, the
mutation is produced in at least one copy of the gene SIBRC1L2. In
another preferred embodiment, the induction of the mutation of step (b)
is performed by chemical mutagens.

[0165] Another aspect of the invention relates to a method for obtaining
potato plants with modified plant architecture, in comparison with the
wild control plant, comprising:

[0166] a) obtaining plant material from a potato plant (parent),

[0167] b) subjecting the plant material of step (a) to a mutagenesis
process c) culturing the mutated plant material until regenerating a
complete plant, and its descendants,

[0168] d) analysing the descendants of the plants of step (c) to detect at
least one mutation in at least one copy the orthologous genes of BRC1
(genes StBRC1L1 and StBRC1L2),

[0169] e) selecting the descendants with at least one mutation in at least
one copy of the genes StBRC1 L1 and StBRC1 L2 which have their plant
architecture modified in comparison with a control type plant,

[0170] f) optionally, culturing the plant selected to obtain descendants
which have said modification of the plant architecture.

[0171] In a preferred embodiment of this aspect of the invention, the
induction of the mutation of step (b) is performed by chemical mutagens.

[0172] Throughout the description and the claims the word "comprises" and
its variants are not intended to exclude other technical characteristics,
additives, components or steps. For persons skilled in the art, other
objects, advantages and characteristics of the invention will be inferred
in part from the description and in part from the practice of the
invention. The following figures and examples are provided by way of
illustration, and are not intended to limit the present invention.

DESCRIPTION OF THE FIGURES

[0173] FIG. 1. Phenotype of the transgenic tomato lines with reduced
activity of the genes SIBRC1L1 and SIBRC1L2. General aspect of the
Moneymaker variety control plant (A) and line 35SCaMV::SIBRC1L1 RNAi (B).
Detail of axillary bud of control plant (C) and of plant 35SCaMV::
SIBRC1L2 RNAi (D). E. General aspect of plants of lines 35SCaMV::
SIBRC1L2 RNAi.

[0175] FIG. 3. Differential expression of the genes StBR1L1 and StBR1L2 in
aerial buds and stolons, quantified by semiquantitative RT-PCR.

[0176] FIG. 4. Phenotype of transgenic potato lines with reduced activity
of StBRC1L1. A. General aspect of Desiree variety control plant (left)
and plant 35SCaMV:: StBRC1L1 RNAi (right). B. Phenotype of aerial
branching of control plants and lines 35SCaMV:: StBRC1L1 RNAi. The x-axis
represents the number of branches. C. Phenotype of underground branching
(stolons) of control plants and lines 35SCaMV:: StBRC1L1 RNAi. The x-axis
represents the number of stolons.

[0177] FIG. 5. Phenotype of stolons of lines 35SCaMV::RNAi StBRC1L1. A.
The transgenic plants (RNAi) produce a greater number of stolons than the
control plants (wt). B. Transgenic plants produce ramified stolons unlike
the control plants.

[0178] FIG. 6. Yield of the transgenic lines with reduced activity of
StBRC1L1. A. Total production of tubers of control individuals (top) and
individuals from independent lines 35SCaMV:: StBRC1L1 RNAi (bottom,
numbered panels). B. Quantification of tuber production. C.
Quantification of the total weight of tubers.

[0179] FIG. 7. Maps of the binary plasmids used for the generation of
lines 35SCaMV:: SIBRC1L1 RNAi and 35SCaMV:: SIBRC1L2 RNAi (left) and
35SCaMV:: StBRC1L1 RNAi and 35SCaMV:: StBRC1L2 RNAi (right). It indicates
the fragments of sense sequence (box A) and antisense sequence (box B)
which are cloned separately by the intron of the pyruvate dehydrogenase
kinase (PDKintron), constituting the RNAi structure. In front of the
sense fragment A there is the 35S promoter of the cauliflower mosaic
virus (35S promoter) and as transcription terminator the octopine
synthase terminator (OCSter). It also indicates the ends of the T-DNA, LB
(left end) and RB (right end) and the position of the gene of neomycin
phosphotransferase, which confers resistance to kanamycin (NPTII KanR).

EXAMPLES

[0180] The invention will be illustrated below by assays performed by the
inventors, which reveal the specificity and efficacy of the modifications
in the expression of the genes SIBRC1L1, SIBRC1L2, StBRC1L1 and StBRC1L2
in the alteration of the plant architecture of the tomato plant and of
the potato plant.

Example 1

Cloning of the Genomic, Promoter and Coding Sequences of the Genes
SIBRC1L1 and SIBRC1L2

[0181] To clone the orthologues to BRC1 of the tomato plant a search was
performed of BRC1-type TCP genes in different databases of solanaceae:
TIGR Solanaceae Genomics Resource BLAST page, TIGR Plant Transcript
Assemblies Database and SOL Genomics Network. To carry out the
comparison, the sequence of amino acids of the TCP box of the BRC1
protein of Arabidopsis was used, and an EST (Expressed Sequence Tags) and
a cDNA was found whose translation gave rise to proteins with high
homology with BRC1 of arabidopsis. The EST EST522935 had 447 bp and the
partial cDNA AY168167, 415 bp. The nucleotide sequences of the genes
SIBRC1L1 and SIBRC1L2 are collected in SEQ ID NO: 7 and SEQ ID NO: 8,
respectively.

[0182] To amplify the complete cDNAs (SEQ ID NO: 15 for SIBRC1L1 and SEQ
ID NO: 16 for SIBRC1L2) of both genes, two different strategies were
followed.

[0183] In the case of SIBRC1L1, two nested primers were designed (Le1, SEQ
ID NO: 17 and Le2, SEQ ID NO: 18) in region 5' of the gene, and the
complete cDNA was amplified with PCR with oligo dT from cDNA of axillary
buds of the tomato plant. In the case of the gene SIBRC1L2, the available
sequence included neither end 5' nor 3', for which reason the cloning was
performed of both ends by the SMART® RACE cDNA Amplification Kit
(Clontech). By using this kit, synthetic adapters were incorporated at
ends 5' and 3' during the synthesis of the cDNA performed from total RNA
of axillary buds of the tomato plant. For the amplification of both ends
using the sequence of the synthetic adapters, two pairs of nested primers
were designed in the available sequence of the gene SIBRC1L2: LeTCP2--F1
(SEQ ID NO: 19) and LeTCP2--F1 nested (SEQ ID NO: 20) for end 3' and
LeTCP2-R1 (SEQ ID NO: 21) and LeTCP2--R1 nested (SEQ ID NO: 22) for end
5'. Once the sequence of both overlapping fragments 5' and 3' were
obtained primers (LeTCP2 cDNA-F, SEQ ID NO: 23) and LeTCP2 cDNA-R, SEQ ID
NO: 24) were designed to amplify the complete gene.

[0184] In the case of the gene SIBRC1L1, two types of cDNA were amplified,
one with an open long-reading phase (1041 pb) and one with an open
short-reading phase (978 pb) by a processing of different introns, whilst
in the case of SIBRC1L2 only one type of cDNA was amplified with an open
reading phase of 1014 pb. The PCR fragments corresponding to the three
cDNAs were cloned in the pGEMT-easy® vector (Promega).

[0185] Once the complete sequence of both genes is known, the fragments
corresponding to their genomic sequence was amplified (SEQ ID NO: 7 for
SIBRC1L1 and SEQ ID NO: 8 for SIBRC1L2), using primers which included
zones 5' and 3' corresponding to each gene: Le1 (SEQ ID NO: 17) and Le3
(SEQ ID NO: 25) to amplify the genomic sequence of SIBRC1L1, and SIBRC1L2
cDNA-F (SEQ ID NO: 23) and LeTCP2 cDNA-R (SEQ ID NO: 24) to amplify that
of SIBRC1L2. In the case of SIBRC1L1, on comparing the genomic sequence
with that corresponding to the coding zone, the existence of two introns
was observed, which were eliminated in the short cDNA, but one of which
is maintained in the long cDNA. In the case of the gene SIBRC1L2, the
comparison of the genomic sequence with the coding showed the existence
of an intron.

[0186] The isolation of the promoter zones of both genes (SEQ ID NO: 5 for
SIBRC1L1 and SEQ ID NO: 6 for SIBRC1L2) was performed using a Genome
Walker® (Clontech) library of tomato. Using this strategy, from
genomic DNA, different Genome Walker® libraries were created by
digestion with different enzymes which produced blunt ends (DraI, EcoRV,
PvuII and SspI) and later linkage of synthetic adapters at the ends
produced by digestion. From nested primers designed at around 100 bp of
the atg of both genes (GSP1-TCP1, GSP2-TCP1--SEQ ID NO: 26 and SEQ ID NO:
27 respectively--for SIBRC1L1 and GSP1-TCP2, GSP2-TCP2--SEQ ID NO: 28 and
SEQ ID NO: 29 respectively--for SIBRC1L2) and of those available for the
adapters, two fragments of 1.kb and 0.7 kb in size were amplified by PCR,
corresponding to the promoting zones of the genes SIBRC1L1 and SIBRC1L2,
respectively. Both fragments were cloned in the pGEMT-easy® vector.

Example 2

Generation of Transgenic Tomato Plants (Solanum Lycopersicum, Moneymaker
Variety) with Loss of Function of the Genes SIBRC1L1 and SIBRC1L2
Silenced by the RNAi Technique

[0187] The DNA fragments chosen to perform the RNA interference are
situated between the TCP box and the R box, highly conserved areas and
characteristic of the TCP genes. Said fragment rings exclusively with the
part of the chosen sequence which guarantees that the silencing is
specific for each gene separately, SIBRC1L1 and SIBRC1L2.

[0188] The fragment used to silence the gene SISRC7L1 has 225 base pairs,
and the sequence is included in SEQ ID NO: 11, and constitutes the
seventh polynucleotide of the invention.

[0189] The fragment used to silence the gene SIBRC1L2 has 415 base pairs,
and the sequence is included in SEQ ID NO: 12, and constitutes the eighth
polynucleotide of the invention.

[0190] Strategy Used for the Generation of the RNAi Constructions for the
Genes SIBRC1L1 and SIBRC1L2.

[0191] To obtain the hairpin structure characteristic of the RNAi, the
fragment selected was cloned in the pHannibal plasmid (CSIRO), which
carried resistance to ampicillin. Said cloning is directed, so that the
fragment enters in direction 5'-3' cloning it with the targets BamH I and
Cla I, and at 3'-5' at the other end of the intron PDK (742 pb), using
the targets Xho I and Kpn I. Therefore, the fragments selected were
amplified using primers containing at end 5' the target sequences for the
different restriction enzymes to be used (the primer for end 5' of
SIBRC1L1 is included in sequence SEQ ID NO: 30, for end 3' of SIBRC1L1 in
sequence SEQ ID NO: 31, for end 5' of SIBRC1L2 in sequence SEQ ID NO: 32
and for end 3' of SIBRC1L2 in sequence SEQ ID NO: 33).

[0192] Once the recombinant plasmids have been obtained with the hairpin
structure of the RNAi, and checked by sequencing, the cassette was
transferred with the pHannibal transgene (3330 pb) cutting with Not I and
it was cloned in the site for the same restriction enzyme of the
BluescriptII SK+ plasmid. In this way, it was possible to leave to one
side of the sequence a Sad site and at the other side a SmaI site, so
that by a digestion with both enzymes of the fragments and of the binary
plasmid pBIN19 (FIG. 7), both fragments were subcloned giving rise to
constructions which have been introduced in the tomato plant (FIG. 7).
The choice was made to use this binary plasmid since it has been
well-established that the pBIN19 plasmid effectively transforms tomato,
giving resistance to kanamycin in bacteria and in plants. The expression
of the transgene is directed by the 35S promoter of the cauliflower
mosaic virus (CaMV35S) (1346 pb) promoting its constitutive expression,
whilst at end 3' of the gene octopine synthase (OCS terminator) (766 pb)
is found of Agrobacterium which acts as transcription terminator. Once
the constructions in Escherichia coli have been obtained, a preparation
was made of the plasmids used to transform Agrobacterium tumefaciens
LBA4404. A single colony was selected from the colonies carrying our
plasmids, which was used to transform to transform tomato plants.

Transformation of Tomato Plants.

[0193] To stably transform tomato plants, the protocol of Ellul et al.
(2003) Theor Appl Genet. 106(2): 231-8.) was used. Following this
protocol, tomato cotyledons were transformed from plants grown in in
vitro conditions in Murashige and Skoog medium with vitamins (Physiol.
Plant. 15:473-497, 1962) supplemented with 2% sucrose. Once the first
true leaves were developed, the cotyledons were cut transversally in one
or two portions (explants), depending on the size, and they were placed
during two days in the dark with the reverse in contact with the
preculture medium (PCM), which includes the hormones AIA and kinetin, at
a final concentration of 4 mg/l. After 48 hours, the explants were
infected by immersing them during 8 minutes in the Agrobacterium culture.
After eliminating the excess Agrobacterium, the explants were placed in
the coculture medium (CCM), which has the same composition of hormones as
the previous, adding acetosyringone. The explants were incubated with the
bacteria during 48 hours in the dark.

[0194] Having concluded the coculture period, the explants were cleaned in
washing medium (WM) plus the antibiotic claforan (500 mg/l) to eliminate
the Agrobacterium, and they were dried on sterile filter paper to pass
them to recovery medium (RM) without selective pressure
(AIA/Kinetin/Claforan). In this medium, they were cultured in light for
two days, after which they were transferred to the first selective medium
(SM) whereto another hormone was added, zeatin (1 mg/l) and the
antibiotic of selection of the transgene kanamycin (50 mg/l). The
explants were cultured in this selective medium until the first change to
fresh medium (with the same composition) after three weeks.

[0195] Calluses were developed from these explants which passed through
four three-week subcultures before developing the first apices.

[0196] Once the apices were well-developed, the calluses were cut and they
were transferred to rooting medium (RM), which includes AIA in low
concentration (0.1 mg/l) to favour root development. Once they were well
developed, the tomato plants were transferred to a mixture of peat and
vermiculite 3:1, maintaining the plants in high humidity conditions
during at least one week to avoid its withering.

[0203] 10 independent transgenic lines were generated of the tomato plant,
Moneymaker variety carriers of the construction 35S::SIBRC1L1 RNAi and
another 10 carriers of the construction 35S::SIBRC1L2 RNAi which were
phenotypically analysed. The T1 individuals indicated that, whilst the
35S::SIBRC1L1 RNAi individuals had a strong apical dominance (they had no
branches), under the same conditions, the 35S::SIBRC1L2 RNAi individuals
had a clear excess of lateral branches in comparison with the wild
branches (FIGS. 1 and 2). These results show that the gene SIBRC1L2 has a
greater importance than SIBRC1L1 in the control of lateral branch growth
in the tomato plant.

Example 3

Cloning of the Genomic, Promoter and Coding Sequences of the Genes
StBRC1L1 and StBRC1L2

[0204] To clone the orthologues to BRC1 of the potato plant a search was
performed of BRC1-type TCP genes in different databases: TIGR Solanaceae
Genomics Resource BLAST page, TIGR Plant Transcript Assemblies Database
and SOL Genomics Network. To carry out the comparison, the sequence of
amino acids of the TCP box of the BRC1 gene of Arabidopsis was used. Two
unigenes were found: TC168465 and TC129597 which were called StBRC1L1 and
StBRC1L2, respectively. Furthermore, knowing the high homology existing
between tomato and potato, and having cloned the SIBRC1L1 tomato gene,
the same primers were tested with genomic potato DNA, for end 5' Le1 (SEQ
ID NO: 17) and Le2 (SEQ ID NO: 18), the latter being a nested primer of
the previous, and Le3 (SEQ ID NO: 25) for end 3'. Based on this sequence
a specific primer was designed (racest1-5\ SEQ ID NO: 34) to localize end
5' of the gene using the PCR-RACE technique with cDNA of axillary buds
and stolons from potato. Based on the sequence obtained, a primer was
designed at end 5': StTCPI-ORF1 (SEQ ID NO: 35). To amplify the cDNA
sequence, a cDNA was used synthesized from the same RNA as for end 5',
but using primer B26 (SEQ ID NO: 36) which includes in its sequence a
polyT tail after the sequence of primer B25 (SEQ ID NO: 37), which makes
it possible to use it as primer of end 3'.

[0206] StBRC1L2 was first partially amplified from the same cDNA used for
the StBRC1L1 gene. Primer B25 was used for end 3', and, for end 5'
primers StTCP2A (SEQ ID NO: 40) and StTCP2B (SEQ ID NO: 41) (nested from
the previous) were used, which had been designed depending on the
sequence of the EST TC129597. From the sequence obtained, end 5' was
localized using PCR-RACE and the specific primers St2-Seq 1 (SEQ ID NO:
42) and the nested one thereof. St2-Seq 2 (SEQ ID NO: 43).

[0207] For the amplification of the complete cDNA, the primers StTCP2-5'
(SEQ ID NO: 44) and B25 were used. The sequence of the cDNA showed a
series of polymorphisms which we consider as two alleles giving rise to
allele 1 and allele 2, as well as to their respective proteins. For the
genomic sequence, primers StTCP2-5' and StTCP2-3' (SEQ ID NO: 45) were
used.

[0208] All amplified PCR fragments corresponding both to parts and all the
sequences of the genes were clones in the pGEMT-easy® vector
(Promega).

Example 4

Generation of Transgenic Potato Plants (Solanum tuberosum, Desiree
Variety) with Loss of Function of the Genes StBRC1L1 and StBRC1L2
Silenced by the RNAi Technique.

[0209] The fragment chosen for the interference of StBRC1L1 is situated
between the TCP box and the R box, zones highly conserved and
characteristic of the TCP genes. Said fragment does not ring with
anything else but this part of the chosen sequence which guarantees a
specific silencing of the StBRC1L1 gene. The fragment has 185 base pairs,
and the sequence is included in the SEQ ID NO: 13, and constitutes the
ninth polynucleotide of the invention.

[0210] The fragment chosen for the interference of StBRC1L2 is also
situated between the TCP box and the R box. Said fragment only hybridizes
with the part of sequence chosen, which guarantees the specific silencing
of the gene StBRC1L2. The fragment has 168 base pairs, is included in the
SEQ ID NO: 14, and constitutes the tenth polynucleotide of the invention.

[0211] Strategy used for the generation of the RNAi constructions for the
genes StBRC1L1 and StBRC1L2.

[0212] To obtain the hairpin structure characteristic of the RNAi, the
fragment selected was cloned in the pHannibal plasmid (CSIRO), which
carried resistance to ampicillin. Said cloning is directed, so that the
fragment enters in direction 5'-3' cloning it with the targets BamH I and
Cla I, and at 3'-5' at the other end of the intron PDK (742 pb), using
the targets Xho I and Kpn I. Therefore, the fragments selected were
amplified using primers containing the target sequences for the different
restriction enzymes to be used at end 5'.

For StBRC1L1

Primer of end 5': (SEQ ID NO: 46)

Primer of end 3': (SEQ ID NO: 47) For StBRC1L2

Primer of end 5': (SEQ ID NO: 48)

Primer of end 3': (SEQ ID NO: 49)

[0213] Once the recombinant plasmid has been obtained with the hairpin
structure of the RNAi, and checked by sequencing, the pHannibal transgene
(3330 pb) was extracted cutting with Not I and it was cloned in the site
for the same restriction enzyme of the binary plasmid pART27 (Gleave,
1992 Plant Mol. Biol. 1992 December; 20(6): 1203-7) (FIG. 7), which
confers resistance to streptomycin and to spectinomycin in bacteria and
to kanamycin in plants. The NotI site of pART27 is localized between the
right and left edges of the plasmid, which guarantees its transfer to the
plant cell on transforming it. The transgene is flanked by the 35S
promoter of the cauliflower mosaic virus (CaMV35S) (1346 pb) for a
constitutive expression and end 3' of the octopine synthase gene (OCS
terminator) (766 pb) of Agrobacterium which acts as transcription
terminator.

[0214] Once the constructions in Escherichia coli have been obtained, a
preparation was made of the plasmids used to transform Agrobacterium
tumefaciens AGLO. A single colony was selected from the colonies carrying
our plasmid, which was used to transform tomato plants.

Transformation of Potato Plants.

[0215] Potato plants were transformed grown in in vitro conditions in
Murashige and Skoog medium with vitamins (Physiol. Plant. 15:473-497,
1962) supplemented with 2% sucrose (MS2). The plants must be between 3
and 4 weeks starting from the time when the plant apex is ringed in fresh
medium.

[0216] The leaves are removed from the plant and the part of the petiole
is eliminated with a scalpel and one or two cuts are made in the central
vein, without these reaching the edges of the leaf. Ten of these leaves
are placed with the top part downwards, in a 9 cm-diameter dish
containing 10 ml of MS2 medium.

[0217] The dish is inoculated with 80 μl of the Agrobacterium culture.
Said culture is initiated at an optical density (OD) at 600 nm of 0.2 in
YEB medium with the suitable antibiotics. When it reaches an OD600
nm of 0.8 it is washed by centrifugation and it is resuspended in the
same volume of YEB medium without antibiotics, which is that used for
inoculation.

[0218] The dishes are incubated during 2 days in the dark, but in the same
temperature and humidity conditions wherein they are going to later grow.
They then pass through a callus induction medium (CIM) maintaining the
position of the leaves with the reverse downwards. After 7-8 days they
are passed to the branch induction medium (BIM). They remain in this
medium until the appearance of calluses and their development on leaves.
The medium is refreshed every 8-10 days.

[0219] When the branches have between 0.5 and 1 cm they are transferred to
the rooting medium consisting of MS medium with 1.6% of glucose, without
any hormone, but with kanamycin (50 mg/L) and claforan (250 mg/L) to
avoid the growth of Agrobacterium.

[0220] After rooting and when the plants have grown, the apex is cut and
transferred to a MS2 medium with kanamycin and claforan in the same
conditions indicated above.

[0221] For growing the plants in the greenhouse, plants which have been in
MS2 medium between 1 and 2 weeks are transferred to receptacles with a
capacity of 50 ml of substrate. The roots are covered with the substrate
and the complete plant is covered with plastic to maintain the humidity,
characteristic condition of in vitro growth. After 3-4 days said cover is
removed.

[0223] In the potato plant there are several types of axillary buds:
aerial buds which give rise to the branches, and the underground buds
which give rise to the stolons, underground stems which tuberize giving
rise to the tubers. The axillary buds of the stolons which are included
in tubers are the tuber eyes.

[0224] The results show that, in the potato plant, StBRC1L1 is expressed
at higher levels in the stolon buds than in the aerial buds, whilst
StBRC1L2 shows an inverse expression template (FIG. 3). This could reveal
the specialization of each orthologue of BRC1 in the control of different
types of buds.

[0225] The phenotype of the lack of function of StBRC1L1 supports its role
in the suppression of the elongation and branching of the stolons. Seven
transgenic lines of 35S::RNAiStBRC1L1 of potato, Desiree variety were
generated, and they were analysed during two generations.

[0226] StBRC1L1 affects both the development of the aerial branches and
that of the stolons since the silenced lines have a greater number of
lateral branches, aerial stolons and underground stolons (FIGS. 4 and 5).

[0227] The underground stolons (which give rise to the tuber) are
branched, unlike the wild stolons which show a strong apical dominance
(FIG. 5). The tuberization time is not affected in these lines. Since
each end of the stolon normally gives rise to a tuber, the high number of
branched stolons makes each plant generate a greater number of tubers
than wild plants (FIGS. 6A and 6B, 64-276.9% more than the controls). In
the experimental conditions wherein the plants were grown (flowerpots of
20 cm diameter) a moderate increase was produced of the yield (17-19%) of
the total weight of the tubers (FIG. 6C). It is very probable that in
optimum conditions the yield is greater with respect to the wild plants.
Furthermore, the lines are fairly vigorous and have delayed senescence.